Studies on calpain expression during differentiation of rat satellite cells in primary cultures in the presence of heparin or a mimic compound

We have synthesized dextran derivatives called RGTAs (for regenerating agen ts) that were designed to mimic some of the properties of heparin or hepara n sulfate to interact with and protect: heparin binding growth factors, Som e of these growth factors have been described to be involved in myogenesis control, In previous studies, we have shown that muscle regeneration in adu lts could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the prese nt study we have used primary cultures of rat satellite cells and treated t hem with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these subs tances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cul tures or myogenic cell lines, have suggested that calpains might be involve d in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the R GTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 mu g/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both sub stances were shown, however, to induce precocious and enhanced differentiat ion of satellite cells. We showed by quantitative RT-PCR analysis that mu-c alpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cul tures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 dur ing differentiation of satellite cells, whereas the level of m-calpain mRNA s was slightly increased at day 12 only, and calpain 3 mRNA was slightly re duced in these differentiating cultures. Interestingly enough, RGTA and hep arin, which both strongly increased differentiation reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in diffe rentiating cultures. These results showed that there was no correlation bet ween the extent of myoblast differentiation and the level of calpain expres sion in satellite cells grown in primary cultures and underscored the diffe rences between these adult cells and fetal myoblasts. (C) 1999 Academic Pre ss.